Nomilin

Six independent plants were used as biological replicates, and about five leaves were sampled from each plant in secondary metabolic profiling. The secondary metabolic profiling was performed by LC-QTOF-MS using a modified method according to Yun et al. [37 (link)]. 100 mg freeze-dried powder was extracted with 80% methanol over night at 4°C. The mixture was centrifuged and filtered. Then, the metabolic profiling were performed using a QTOF 6520 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) coupled to a 1200 series Rapid Resolution HPLC system.
The raw data was processed by Agilent Mass Hunter Qualitative Analysis (version B. 04.00, Aglient Technologies) and Mass Profiler Software (version B.02.02, Aglient Technologies). Then PCA analysis was performed using the filtered and normalized data. Metabolite identification was carried out by comparing mass spectra and retention time with those of authentic standards. Nine authentic standards, namely, narirutin, naringin, hesperidin, neohesperidin, didymin, sinensetin, limonin, nomilin, and nobiletin were obtained from Sigma Chemical Co. (St. Louis, MO).

HPLC-grade ethanol, methanol, dimethyl sulfoxide (DMSO) and methyl tert-butyl ether (MTBE) were purchased from Tedia (Fairfield, OH, USA). Deionized water (18.20 MΩ cm) was produced with a Milli-Q system from Millipore (Bedford, MA, USA). Feruloyl putrescine, β-cryptoxantin, α-carotene, β-carotene, lutein, lycopene, 9-Z-violaxanthin, limonin, nomilin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Feruloyl galactaric acid, vicenin-2, narirutin-4′-glucoside, hesperetin glucoside, isovitexin, vitexin, narirutin, diosmin, hesperidin, isosakuranetin rutinoside, sinensetin, nobiletin, tetramethylscuttelarien, heptamethoxyflavone, tangeretin, and 5-desmethyl nobiletin were obtained from the USDA (United States Department of Agriculture) citrus flavonoid collection from previous studies [32 (link),33 (link),34 ].